The aim of this project is to determine the functional and structural properties of the hearing associated protein otoferlin. This protein is believed to directly regulate membrane fusion and the resultant neurotransmitter release from inner hair cells in the cochlea. Mutations in otoferlin result in profound hearing loss at birth. However, the exact role of otoferlin in hearing is not well understood. The following specific aims pursued under this award are intended to improve our understanding of otoferlin in the auditory pathway. If otoferlin is indeed directly involved in exocytosis, it should posses certain characteristic functional properties which allow it to partake in membrane fusion events. In the first aim, the hypothesized ability of otoferlin to bind SNARE proteins and accelerate membrane fusion will be directly tested for the first time using an in vitro fusion assay. This will establish a functional role for otoferlin in the exocytosis pathway. The calcium binding properties of otoferlin will also be probed in an attempt to determine whether the protein is a calcium sensor. The second aim will elucidate the structure of the protein using X-ray crystallographic methods so as to provide a structural basis for the observed functional properties. Several pathological mutant forms of the protein will also be examined so as to determine the molecular basis for the pathology. Third, the relationship between otoferlin and lipid membranes will be elucidated. Manipulation of lipid bilayers is a common characteristic of proteins involved in membrane fusion and exocytosis. Yet it is currently unknown whether otoferlin can indeed bind to and alter membrane morphology. This aim will test the hypothesis that otoferlin can penetrate and induce curvature in lipid bilayers in a calcium dependent manner as a mechanistic step in accelerating membrane fusion. PUBLIC HEALTH RELEVANCE: This proposal seeks to understand the role that the protein otoferlin plays in hearing and why mutations in this protein result in deafness. Both functional and structural studies will be undertaken to provide a better understanding as to how this cochlea-specific protein is able to control neurotransmitter release.